An outbreak of acute diarrhoeal disease caused by Shigella sonnei in a village in Dibrugarh district, Assam

Study setting and location

Five acute diarrhoeal cases were admitted to Assam Medical College from Ougurigaon village in Moran area of Dibrugarh district, Assam from June 8-9, 2022, with a history of food consumption at a birthday party on June 7, 2022, before the episode. The admitted cases were suspected to be part of an acute diarrhoeal disease outbreak. All admitted ADD cases who attended the birthday party were interviewed on June 9, 2022, to confirm the occurrence of food-borne outbreak. In this episode, we defined the case of ADD including acute gastroenteritis (AGE) as any affected individual who presented with complaints of passage of three or more episodes of loose or liquid stools in last 24 h with or without dehydration within three days of eating at the birthday party as per the case definition of IDSP p-form^9,10.

Epidemiological study

On June 14, a retrospective cohort study was carried out on individuals attending the above-mentioned birthday party held on June 7, 2022. An interdisciplinary team comprising an epidemiologist, a medical microbiologist, a biological scientist, field workers, a laboratory technician, a data entry operator and ASHA (Accredited Social Health Activist) workers from the village visited the households. The kitchen, where the birthday party food was prepared, was examined, and the residences of those who attended the event were also surveyed. Sociodemographic information such as age and gender, along with details about the birthday party, including the source of foods, a list of prepared items, information about food handlers, food exposure, timing of food consumption, illness details (onset date and time), signs and symptoms, interventions and outcomes were collected using a pre-designed outbreak investigation form (Supplementary Material). For statistical analysis, Statistical Package for Social Sciences (SPSS) Statistical Software Version 14.0 (IBM corp., Armonk, NY, USA) was used for data management and analytics. The spot map and the line list were prepared, and the epidemic curve was plotted by the date and time of the onset of ADD along with the calculation of age- and gender-specific attack rates. Univariate analysis was carried out using the variables such as age groups, gender and food items [cake, rice and daal (lentils), egg curry, chicken curry, chicken intestine fry, pork fry and fish fry]. The P value <0.05 was considered as statistically significant. The association measures were calculated by odds ratios (OR; unadjusted and adjusted) and their 95% confidence interval (CI) using the logistic regression method. For calculating the attributable fraction, the sum of the category-specific differences between the total number of observed cases and the total number of expected cases was divided by the sum of the observed cases.

Laboratory investigations

Thirteen rectal swabs were saved in Cary Blair Medium, and stool samples were collected using sterile cotton swabs from the affected individuals and were examined at the Microbiology and Immunology Laboratory of ICMR-RMRC, Dibrugarh within 3 h of collection^11,12. Briefly, stools were cultured on MacConkey, Xylose-lysine deoxycholate (XLD), and Hektoen enteric (HE) agars (HiMedia Laboratories, Mumbai) to isolate Escherichia coli, Shigella spp., and Salmonella spp., respectively. Sucrose-Xylose-lactose non-fermenting red colonies on XLD agar with green colour colonies on Hektoen Enteric agar were suspected as Shigella spp., were further tested biochemically for differentiating between different strains^11,13. Antimicrobial susceptibility was tested using commercial antibiotic discs (HiMedia Laboratories, Mumbai) and the inhibition zone diameters were interpreted as resistant to a particular antimicrobial agent as per standard CLSI guidelines¹².

Polymerase chain reaction (PCR) assay

The DNA was isolated from the S. sonnei isolates using a Bacterial DNA isolation kit (HiPure Genomic DNA Purification Kit, HiMedia Laboratories, Mumbai). PCR was performed using virulence genes of EIEC pathotype for virF and ipaH genes using a simplex PCR¹⁴. For amplification of the virF gene Forward 5’ AGC TCA GGC AAT GAA ACT TTG AC 3’ primer and Reverse 5’ TGG GCT TGA TAT TCC GAT AAG TC 3’ primer was used that amplifies a product of 618 bp. For the ipaH gene Forward 5’CTC GGC ACG TTT TAA TAG TCT GG 3’ and Reverse 5’ GTG GAG AGC TGA AGT TTC TCT GC 3’ primers were used to amplify a PCR product of 933 bp. The thermal profile used for PCR was initial denaturation at 94°C for 5 min, samples were amplified for 35 cycles, with each cycle consisting of 1.5 min at 94°C for denaturation, 1.5 min at 60°C for primer annealing and 1.5 min at 72°C for strand elongation and a final elongation at 72°C for 7 min. The PCR products were visualized in 1.5% agarose gel stained with ethidium bromide. However, genomic sequencing was not done. Confirmation of the identified pathogen was done by the ICMR-National Institute of Cholera and Enteric Diseases, Kolkata using biochemical tests and serotyping for identification of Shigella spp.

Environment investigation

The sole food handler and the cook was one – the father of the birthday child. He was interviewed about the recent illness, and a rectal swab was collected for analysis. Borewell groundwater samples were collected from the household that hosted the event for laboratory testing as per the standard operating procedures (SOP)¹¹.

Descriptive and analytical epidemiology

The index case was a 14 yr old boy, who was admitted to the department of Medicine, Assam Medical College, Dibrugarh on June 8, 2022, with chief complaints of severe vomiting, bloody dysentery, high fever, nausea, and loss of consciousness resulting in a fall in the bathroom. The affected boy was severely dehydrated during admission due to the passage of loose, watery stools and required administration of intravenous IV fluids and antibiotics. Interviews with both the affected boy and the guardian revealed that the boy had attended the Ougurigaon birthday party before experiencing illness. We recorded through further investigation that 41 people had attended the birthday party on June 7, 2022, at Ougurigaon village. The birthday celebration event including food consumption lasted from 12:30 PM to 4:30 PM, following which 25 people developed ADD [overall attack rate 60.04% (25/41)]. The family members of the host were also affected, and one required hospitalization. Among the affected individuals all had weakness, 19 developed fever, 18 abdominal cramps and 17 cases had vomiting. The mean incubation period for the onset of symptoms from the time of food consumption was 26.36±8.76 h (±SD) and indicated a common source of contamination with no secondary cases. The last case was reported on June 10, 2022, at 9:00 AM as shown in the Epidemic curve (Figure). Two outlier cases were also traced five days post-cessation of outbreak, though they did not attend the event, but they had consumed only chicken curry on June 8, 2022, that was sent to them by the host on June 7, 2022 and fell ill on June 10, 2022. However, no chicken intestine fry was sent to them by the host. Table I shows the age and gender distribution among ill and non-ill persons. The highest attack rate (AR; 100%) was in the age group 0-4 yr followed by AR of 66.7 per cent in the age group 40-64 yr. The lowest AR was 50 per cent in the 25-39 yr age group (Table I). Females had higher rate of infection (64%) compared to males; no case fatality was reported. Table I also presents the distribution of the explanatory variables among the 16 people who did not fall ill after attending the event.

Close

Figure.

Epidemic curve of the outbreak from exposure in the event and the appearance of symptoms of Acute Gastrointestinal Disease (AGE) among those who were affected during the outbreak. ADD, acute diarrhoeal disease.

Export to PPT

The food served at the birthday party was prepared by the father of the birthday child and consisted of cake, rice, daal (lentils), egg curry, chicken (broiler) curry, fried chicken intestine, fried pork and fried fish. The fish was cut and dressed by a vendor in the nearby market. Moreover, he bought 10-12 live broiler chickens from a local retailer, which was relatively smaller in size. Family members mentioned that the chickens appeared unwell, exhibiting signs of apparent illness- such as being underweight, having ruffled feathers, and showing exposed skin patches. He had culled all the chickens, eviscerated the intestines and prepared the chicken curry and fried intestines with red gram, onion and green chilli flakes. As per the host, the food was kept on the serving tables covered but not kept under refrigeration as all the food items were to be consumed within the same day. There was no leftover food in the party. Further, both parents did not suffer from a similar illness before the event.

Out of the eight food items served at the party, only one food item was significantly associated with ADD: chicken (broiler) curry, which was consumed by the 30 individuals of which 76.6 per cent (23) developed ADD. Thirteen people consumed chicken intestine, of which nine (69.2%) developed ADD. However, it was not significantly associated with the illness (Table I). The OR was statistically significant for exposure to chicken curry consumption and occurrence of ADD (OR=14.8; 95% CI: 2.75-85.11); P<0.05. The analysis of the food-specific population attributable fraction (PAF) was highest for chicken curry 70.18 per cent as shown in the Table I. All the other food items were not significantly associated with the occurrence of ADD. Further, the logistic regression was performed to explore the effects of gender, age groups, food items consumed and occurrence of ADD in the cohort. The Model summary of the logistic regression showed Nagelkerke R² value of 0.714 showing good fit predicting 71.4 per cent of the variance in the ADD cases and correctly classified 71 per cent of the cases. The consumption of chicken curry (OR=43.89; CI: 1.83-1054.35); P<0.05 was significantly associated with occurrence of ADD. The association of other independent variables such as gender, age groups, food items consumed at the party (cake, rice and daal, egg curry, chicken intestine fry, pork fry and fish fry) and occurrence of ADD was not statistically significant (Table II).

Laboratory confirmation

The physical examination of the stool showed presence of mucous (100%) and blood (12). Thirteen samples tested positive for S. sonnei as the causative pathogen and no other enteric pathogen was detected. The water sample collected from the borewell was found satisfactory. S. sonnei isolated from the cases showed highest resistant to nalidixic acid (71.42%), ceftazidime, ceftriaxone and cefotaxime (64.28%). The high sensitivity was seen for gentamicin (64.28%), chloramphenicol (57.14 %) and least resistance to chloramphenicol (14.28%), gentamicin (28.57%), cefoxitin (42.86%) followed by azithromycin (50%) and tetracycline (50%). The PCR results for all S. sonnei isolates were positive for virulence encoding regions of the invasion plasmid antigen H (ipaH) gene.

Environmental findings

The hygienic condition of the room where the food was prepared and stored seemed satisfactory to the investigators with no apparent sight of flies or unclean areas nearby. The hygienic practices of the food handlers were found satisfactory as nails were trimmed adequately. Also, all the family members followed safe hand-washing practices. The investigators also inspected the overall sanitary conditions in the household, which were satisfactory- with separate toilet, no mixed dwelling and availability of bore-well in the house. The borewell groundwater samples on laboratory testing and analysis were found satisfactory for drinking, and no family members fell ill by drinking that water before or after the event.